摘要
目的 探讨氯胺酮通过诱导B103细胞自噬产生抗帕金森病 (Parkinson's disease, PD)的作用及分子机制。 方法 以B103细胞为实验模型,分别以不同浓度氯胺酮(0、100、200、400 μmol/L)及400 μmol/L氯胺酮+5 mmol/L 3-methyladenine(3-MA)处理后,采用噻唑蓝[3-(4,5-dimenthyl-2-thiazolyl-2,5-diphenyl-2-H-tetrazolium bromide, MTT)]染色法检测细胞增殖率;应用吖啶橙与Lyso-Tracker Red染色,荧光显微镜检测细胞自噬;Western blot检测相关蛋白Beclin-1、微管相关蛋白1轻链3(microtubulesas sociated protein light, LC3)、蛋白激酶B(protein kinase B, Akt)、p70S6K、雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)、α-synuclein(α-syn)及β-synuclein(β-syn)蛋白的表达。 结果 不同浓度氯胺酮作用于神经母细胞瘤B103细胞7 d后,MTT染色法检测结果显示,100、200、400 μmol/L分别与0 μmol/L比较,细胞增殖抑制率差异无统计学意义(P〉0.05);吖啶橙与Lyso-Tracker Red染色结果提示,随着氯胺酮浓度增加,自噬作用逐渐增强(P〈0.05);Western blot结果显示,400 μmol/L较0 μmol/L自噬相关蛋白LC3-Ⅰ、磷酸化(phospho, p)-Akt、p-p70S6K以及 p-mTOR表达减少,LC3-Ⅱ、Beclin-1表达增多(P〈0.05);此外,400 μmol/L较0 μmol/L表达PD标志性蛋白α-syn减少而β-syn增多(P〈0.05)。自噬抑制剂(3-MA,5 mmol/L)预处理可逆转氯胺酮对B103细胞的上述作用(400 μmol/L+3-MA与400 μmol/L比较,P〈0.05)。 结论 氯胺酮通过抑制Akt/mTOR信号转导通路诱导B103细胞自噬并降解α-syn从而产生抗PD的作用。
Objective To investigate the effect and molecular mechanism of ketamine on anti-Parkinson's disease(PD) on rat B103 neuroblastoma cells. Methods B103 cells were used as the experimental models. After the treatment with ketamine at the concentration of 0, 100, 200, 400 μmol/L and 400 μmol/L ketamine+5 mmol/L 3-methyladenine (3-MA), cell proliferation effects of ketamine were detected using 3-(4,5-dimenthyl-2-thiazolyl-2,5-diphenyl-2-H-tetrazolium bromide(MTT) assay. Autophagy was detected with acridine orange staining and Lyso-Tracker Red staining using fluorescence microscopy. Expression of the proteins associated with Beclin-1, microtubulesas sociated protein light(LC3), protein kinase B(Akt), p70S6K, mammalian target of rapamycin(mTOR), α-synuclein (α-syn) and β-synuclein (β-syn) was evaluated using Western blot. Results The proliferation of B103 cells had no significant influence after treatment with ketamine (100, 200, 400 μmol/L) vs 0 μmol/L for 7 d (P〉0.05). Acridine orange staining and Lyso-Tracker Red staining showed that ketamine triggered B103 cell autophagy in a dose-dependent manner (P〈0.05). Western blot results showed that the expression levels of LC3-Ⅰ, phospho(p)-Akt, p-p70S6K and p-mTOR were lower in 400 μmol/L than the levels in 0 μmol/L. The expression levels of LC3-Ⅱand Beclin-1 were higher in 400 μmol/L than expression levels in 0 μmol/L (P〈0.05). Expression levels of Parkinson's disease marker protein α-syn decreased and β-syn were higher in 400 μmol/L than 0 μmol/L (P〈0.05). After pretreatment with 3-MA(5 mmol/L), the anti-PD effect of ketamine on B103 cells was reversed (400 μmol/L+3-MA and 400 μmol/L, P〈0.05). Conclusions Ketamine exerts anti-PD effect by inhibiting Akt/mTOR signaling pathway, inducing autophagy in B103 cells and degrading α-syn.
作者
王莹
宋俊杰
慎晓飞
王丹丹
洪道先
Wang Ying;Song Junjie;Shen Xiaofei;Wang Dandan;Hong Daoxian(Department of Anesthesiology, the First Affiliated Hospital of Henan University of Medicine, Kaifeng 475001, China)
出处
《国际麻醉学与复苏杂志》
CAS
2018年第5期390-394,I0001,共6页
International Journal of Anesthesiology and Resuscitation
基金
国家自然科学基金(81600940)
河南省科技发展计划项目(142102310246)
关键词
氯胺酮
帕金森病
自噬
蛋白激酶B/雷帕霉素靶蛋白信号转导通路
Ketamine
Parkinson's disease
Autophagy
Protein kinase B/mammalian target of rapamycin signaling transduction pathway